Casein kinase II promotes piRNA production through direct phosphorylation of USTC component TOFU-4

Piwi-interacting RNAs (piRNAs) are genomically encoded small RNAs that engage Piwi Argonaute proteins to direct mRNA surveillance and transposon silencing. Despite advances in understanding piRNA pathways and functions, how the production of piRNA is regulated remains elusive. Here, using a genetic screen, we identify casein kinase II (CK2) as a factor required for piRNA pathway function. We show that CK2 is required for the localization of PRG-1 and for the proper localization of several factors that comprise the ‘upstream sequence transcription complex’ (USTC), which is required for piRNA transcription. Loss of CK2 impairs piRNA levels suggesting that CK2 promotes USTC function. We identify the USTC component twenty-one-U fouled-up 4 (TOFU-4) as a direct substrate for CK2. Our findings suggest that phosphorylation of TOFU-4 by CK2 promotes the assembly of USTC and piRNA transcription. Notably, during the aging process, CK2 activity declines, resulting in the disassembly of USTC, decreased piRNA production, and defects in piRNA-mediated gene silencing, including transposons silencing. These findings highlight the significance of posttranslational modification in regulating piRNA biogenesis and its implications for the aging process. Overall, our study provides compelling evidence for the involvement of a posttranslational modification mechanism in the regulation of piRNA biogenesis.

Overall, this is a very nice manuscript, that bring to light the PTM-aspects of the piRNA pathway in C. elegans.This is uncharted territory, and the reported findings make a strong case for the proposed model.I support publicafion in Nature Comm., but do think that the authors should improve a number of aspects of their work before the work can really be accepted for publicafion.
Main points: 1.The authors successfully idenfified potenfial TOFU-4 phosphorylafion sites and supported their findings with in vitro data.However, to strengthen their conclusions, it is essenfial for the authors to demonstrate the difference in phosphorylafion levels between wild-type TOFU-4 and the TOFU-4 (S131A S229A) mutant in vivo, for example, by phos-tag gels.This will help account for other factors that may influence phosphorylafion (is TOFU-4 possibly phosphorylated at other sites?) 2. The authors observed that KIN-3 coimmunoprecipitated TOFU-4 and according to the previous Y2H data, this interacfion is direct.To test whether this interacfion may be responsible for TOFU-4 phosphorylafion, it is important to test if the TOFU-4 (S131A S229A) mutant can sfill interact with KIN-3 or not.Does the observed interacfion reflect substrate binding (in which case it should be lost) or another mode of interacfion?
3. The study shows that TOFU-4 (S131A S229A) has reduced co-precipitafion with PRDE-1 compared to wild-type TOFU-4.To strengthen the idea that TOFU-4 phosphorylafion status drives this interacfion, the authors should invesfigate the effect of the phosphomimefic mutant TOFU-4 (S131D S229D) on coprecipitafion with PRDE-1.Also Y2H tesfing of such mutants would be interesfing to report.4. The authors refer to the C. elegans phosphoproteome dataset that used a mixed worm populafion for the phosphoproteome.The current study should ideally show the changes in phosphorylafion level of USTC proteins after kin-10 and kin-3 deplefion using (for instance) phos-tag gels (in young adults).

5.
It would be beneficial to include mass-spectrum data for TOFU-4 (S131A S229A) coimmunoprecipitafion.This could provide a broader view of the interacfions involved and offer more insights into the specificity of the observed interacfion (changes).
6.When reporfing on sequencing, the authors write that 2 samples were sequenced.Yet, their plots show error bars.However, no stafisfics are possible with n=2.Plofting both replicates next to each other would be the proper way to show the data.Befter would be to take n>2 and do proper stafisfics.However, with the strong effects reported, this could be considered unnecessary.In case the error bars reflect the analysis of the many different piRNA loci this is also not correct, as the error bar would not reflect on experimental variafion but on variability of expression of individual piRNA loci (which is not the aim of the study).

7.
Precursor and mature piRNA sequencing should be done for the phosphorylafion mutants, and the ageing.Sensor acfivafion could also have a different cause, and sequencing would address that concern.8.In general, the imaging of any situafion is restricted to a single image.No sense of variability can be deduced from this.Some form of quanfificafion has to be presented.9. How does KIN-3:GFP look in tofu-4 mutants?Does it go away from eth presumed chromafin?Is there any colocalizafion between KIN-3 and PRDE-1?10.In the phospho mimic mutant that was made both serines were mutated.However, there is no data to show that this is the relevant situafion.Single mutafions should be tested before anything can be concluded.The double mimic could in some unforeseen way erase the effect of single events.Is the phospho mimic in PRDE-1 foci?

Minor:
Please annotate the pachytene zone in 1A,B.For non-elegans researchers the presented informafion is too scarce.
Please provide reference for line2h and turmoil1 acfivafion in piRNA mutants (page 5, boftom) Please show the recombinant proteins purified from E. coli.The readers need to know about the quality of the protein preps used.
In the IP-Westerns, please provide informafion about quanfifies (how much was used for input in relafion to the IP samples?) Tesfing CK2 acfivity in extracts seems a bit of a crude assay, given that many more kinases exist.Can a control be provided using extract from a kin-3 mutant (using the AID system that the authors developed)?
Page 13: '…promotes the subnuclear localizafion…' It is unclear to me what is meant.Not much is known about the subnuclear localizafion of PRDE-1 foci, apart from that there are foci.So what can be 'promoted'?I giess the author aim at potenfial colocalizafion of these foci with nuclear pores?Please explain befter what is meant.
We would like to thank the reviewers for their insightful and constructive feedback.Using these comments as a guide, we have revised our manuscript.Our revisions encompass the integration of new data and we have made extensive revisions to both the results and the discussion sections.These changes include performing the following additional experiments:  We performed phos-tag gel experiments to detect the phosphorylation (supplementary Figure 4a). We conducted the pull-down assay to examine whether TOFU-4 (S131A S229A) mutant retains its ability to interact with KIN-3 (supplementary Figure 4d). We conducted the co-immunoprecipitation (co-IP) assay to compare the interaction capacity between the wild type TOFU-4 and the phospho mimetic mutant TOFU-4 (S131D S229D), and also we checked the localization of PRDE-1 foci in wild type and tofu-4 (S131D S229D) mutants (Figure 5b-e). We performed the small RNA sequencing to detect the precursor and mature piRNA in phosphorylation mutants and in aging (Figure 4f, g and Figure 6g,  h). We examined KIN-3:GFP localization in tofu-4 mutants and the colocalization between KIN-3 and PRDE-1 (supplementary Figure 2d, e).Here are our point-by-point responses to the reviewers' comments.

Zhang et al. reports the casein kinase II (CK2) as a novel factor for the assembly of the upstream sequence transcription complex (USTC) and piRNA transcription. Using a reverse genetic approach, the authors identify that CK2 is required for piRNA-mediated silencing. CK2 promotes PIWI protein PRG-1 expression and USTC piRNA focus formation. Previous studies reported that CK2 mediates protein phosphorylation to affect protein-protein interaction. Using co-IP (in vivo) and phosphorylation assays (in vitro), the authors investigate that CK2 interacts with TOFU-4 and specifically phosphorylates TOFU-4 to promote the assembly of USTC. Meanwhile, Figure 6 proves that CK2 affects PRG-1 and USTC in the aging process. This paper is a very complete analysis of how CK2 participates in piRNA gene transcription. I recommend publication in current form. Besides I have several minor queries.
We thank the reviewer for the positive comments on our work.

1) In Figure 3B, why free GFP became prominent when there is an intact GFP::PRG-1 translation fusion?
We apologize for not explaining this result better.We have revised the paragraph on this finding as follows to address the reviewer's question: "Previous studies have shown that both PRG-1 protein levels and its localization to peri-nuclear nuage, P granules, are reduced in mutants that disrupt expression of piRNA precursors, while PRG-1 mRNA levels remain unaffected 1 .These findings suggest that PRG-1 like some other Argonaute proteins such as Ago2 becomes unstable when unloaded 2 .Consistent with these previous findings we found that in kin-3 depleted animals, PRG-1 mRNA levels were not reduced compared to wild type (Supplementary Fig. 2a), while in contrast, GFP::PRG-1 failed to localize in P-granules and instead localized diffusely in the cytosol (Fig. 3a).We observed an identical change in GFP::PRG-1 localization in animals with mutations in tofu-4 (Fig. 3a), a gene previously shown to be required for piRNA transcription and for PRG-1 protein stability 3 .When assayed by western blotting with GFP-specific antibodies in kin-3 and tofu-4 mutants, the band corresponding to full-length GFP::PRG-1 protein was strongly reduced and a 30KD band, corresponding in size to free GFP, became prominent (Fig. 3b).This finding suggests that the N-terminal GFP tag is proteolytically released from GFP::PRG-1 during the PRG-1 instability induced by kin-3 and tofu-4 mutations, and suggest that persistence of this proteolytic fragment explains the cytoplasmic GFP fluorescence observed in the mutants." 2) TOFU-4 is a component of the USTC complex and KIN-3 was identified as a yeast two-hybrid (Y2H) binding partner of TOFU-4.We wonder whether there are other unidentified USTC factors or UAD-2 or other integrator factors also be targeted by CK2?
We have modified the discussion to address this possibility: Discussion Pages 13: "A search within the phosphorylation database 4 focusing on the USTC factors PRDE-1, SNPC-4, TOFU-4 and TOFU-5, in addition to the recently identified chromodomain factor UAD-2 which localizes to piRNA cluster chromatin 5 , revealed that among these factors, TOFU-4 is the sole protein displaying detectable phosphorylation.While there could be additional piRNA biogenesis factors like UAD-2 subject to CK2 phosphorylation, our study suggests that posttranslational modification of TOFU-4 by CK2 is important for assembly of the piRNA biogenesis machinery and for piRNA pathway funciton." 3) The RNAi screen was performed against all 947 genes in the embryo lethal subset of the C. elegans RNAi collection (Ahringer).A list of the 947 genes or a citation of the list should be included.
As suggested by the reviewer, we have incorporated the list of the 945 tested genes into the updated manuscript (Table 1).

Reviewer #2 (Remarks to the Author):
The manuscript by Zhang et al addresses the role in CK2 (of which KIN-3 is a key subunit in C. elegans) in the worm piRNA pathway.They find that TOFU-4, a subunit of a transcription complex that acts at piRNA loci, is a substrate of CK2 and that phosphorylation of TOFU-4 helps the formation of the typical transcription foci at piRNA loci, and is important for silencing a piRNA sensor.They also show that during ageing TOFU-4 phosphorylation drops, in parallel to a decline of CK2 activity, and accompanied by loss of piRNA silencing efficiency.
Overall, this is a very nice manuscript, that bring to light the PTM-aspects of the piRNA pathway in C. elegans.This is uncharted territory, and the reported findings make a strong case for the proposed model.I support publication in Nature Comm., but do think that the authors should improve a number of aspects of their work before the work can really be accepted for publication.
We thank the reviewer for the thoughtful and constructive comments.

Main points: 1. The authors successfully identified potential TOFU-4 phosphorylation sites and supported their findings with in vitro data. However, to strengthen their conclusions, it is essential for the authors to demonstrate the difference in phosphorylation levels between wild-type TOFU-4 and the TOFU-4 (S131A S229A) mutant in vivo, for example, by phos-tag gels. This will help account for other factors that may influence phosphorylation (is TOFU-4 possibly phosphorylated at other sites?)
We thank the reviewer for this suggestion, we performed phos-tag analysis on TOFU-4 but no mobility shift was detected, even on protein phosphorylated by CK2 in vitro.Phosphorylation does not always lead to a change in protein mobility 6 .We have added the following to the results section to address these new studies: "In some instances phosphorylation of a protein can cause a shift in mobility on an SDS gel 6 .To ask if phosphorylation of TOFU-4 by CK2 in vitro, or in vivo alters TOFU-4 mobility we used the previously published phos-tag gel method 7 .However, no significant shift was observed even for the in vitro phosphorylated TOFU-4 (Supplementary Fig. 4a)."

The authors observed that KIN-3 coimmunoprecipitated TOFU-4 and according to the previous Y2H data, this interaction is direct. To test whether this interaction may be responsible for TOFU-4 phosphorylation, it is important to test if the TOFU-4 (S131A S229A) mutant can still interact with KIN-3 or not. Does the observed interaction reflect substrate binding (in which case it should be lost) or another mode of interaction?
As suggested by the reviewer, we conducted the pull-down assay to examine whether TOFU-4 (S131A S229A) mutant retains its ability to interact with KIN-3.We found that TOFU-4 (S131A S229A) still interacts with KIN-3.Consequently, we think that CK2 phosphorylation of TOFU-4 does not influence the binding between TOFU-4 and KIN-3, but rather affects the assembly of PRDE-1 foci.The results have been included in the supplementary Figure 4d and are discussed on Page 13 as follows: "Mechanistically, our findings suggest that CK2 functions upstream of USTC assembly.TOFU-4 is a direct CK2 substrate and mutation of presumptive CK2 phosphorylation sites in TOFU-4 did not affect its interaction with KIN-3 (Supplementary Fig. 4d) but instead reduced the association of TOFU-4 with the USTC component PRDE-1."

The study shows that TOFU-4 (S131A S229A) has reduced co-precipitation with PRDE-1 compared to wild-type TOFU-4. To strengthen the idea that TOFU-4 phosphorylation status drives this interaction, the authors should investigate the effect of the phosphomimetic mutant TOFU-4 (S131D S229D) on co-precipitation with PRDE-1. Also Y2H testing of such mutants would be interesting to report.
We thank the review for these suggestions.We conducted the in vivo studies.Our results suggested that, while the alanine substitutions abolished the interaction, the phospho mimetic mutant TOFU-4 (S131D S229D) still exhibited co-precipitation with PRDE-1.We noted, however, a 40% reduction in the co-IP, suggesting that the aspartic acid residues do not fully mimic phosphorylation, a finding consistent with the intermediate effect on silencing (Supplementary Fig. 4b).Moreover, consistent with these findings we now include localization studies showing that compared to the wild type protein, TOFU-4 (S131D S229D) protein is still co-localizated with PRDE-1 foci, albeit with smaller foci.These results have been included in the main Figure 5b-e.

The authors refer to the C. elegans phosphoproteome dataset that used a mixed worm population for the phosphoproteome. The current study should ideally show the changes in phosphorylation level of USTC proteins after kin-10 and kin-3 depletion using (for instance) phos-tag gels (in young adults).
This has been done.See our response to question 1.

It would be beneficial to include mass-spectrum data for TOFU-4 (S131A S229A) co-immunoprecipitation. This could provide a broader view of the interactions involved and offer more insights into the specificity of the observed interaction (changes).
We thank the reviewer for this valuable suggestion which could no doubt lead to many future lines of study.While time and resources do not permit a full-scale proteomic study on TOFU-4 we have taken a candidate approach to broaden our understanding of how TOFU-4 (S131A S229A) impacts the co-assembly of other USTC factors.In line with our results, we found that SNPC-4 foci are disrupted in tofu-4(S131A S229A) mutants as well.
6.When reporting on sequencing, the authors write that 2 samples were sequenced.Yet, their plots show error bars.However, no statistics are possible with n=2.Plotting both replicates next to each other would be the proper way to show the data.Better would be to take n>2 and do proper statistics.However, with the strong effects reported, this could be considered unnecessary.In case the error bars reflect the analysis of the many different piRNA loci this is also not correct, as the error bar would not reflect on experimental variation but on variability of expression of individual piRNA loci (which is not the aim of the study).
We apologize for our careless use of the error bars.We have corrected this.These results have been included in main Figure 2b, c and supplementary Figure 1.

7.
Precursor and mature piRNA sequencing should be done for the phosphorylation mutants, and the ageing.Sensor activation could also have a different cause, and sequencing would address that concern.
As suggested by the reviewer, we performed the small RNA sequencing.Consistent with our sensor reporter, we found that in phosphorylation mutants and in aging, both the precursor and mature piRNA are deceased.These results have been included in the main Figure 4f, g and Figure 6g, h.
Results Page 10: "To ask if CK2 mediated phosphorylation of TOFU-4 affects piRNA production, we performed small RNA sequencing.We observed that the levels of both mature piRNAs and of piRNA precursors were reduced in the tofu-4(S131A S229A) mutants (Fig. 4f, g)." Results Page 12: "Consistently, we found the mature piRNA and the piRNA precursor levels were reduced in the aging process (Fig. 6g, h)." 8.In general, the imaging of any situation is restricted to a single image.No sense of variability can be deduced from this.Some form of quantification has to be presented.
We have clearly stated the number and percentage in the paper.Specifically, for Figures 3a, 3c, 3d, 3e, 5c, 5d and 5e, more than 50 worms are counted, the phenotype is similar.For Figure 6, the number and percentage are labeled in the figure.

How does KIN-3:GFP look in tofu-4 mutants? Does it go away from eth presumed chromatin? Is there any colocalization between KIN-3 and PRDE-1?
As suggested by the reviewer, we examined KIN-3:GFP localization in tofu-4 mutants, but did not observe significant change.Since KIN-3:GFP is widely expressed in the cell, we can see the colocalization between KIN-3 and PRDE-1.However, we did not detect any enriched signal of KIN-3::GFP in PRDE-1 foci.The results have been included in the supplementary Figure 2d, e.
Results Page 8: "Indeed, we can see the colocalization between KIN-3 and PRDE-1.However, we did not detect any enriched signal of GFP::KIN-3 in PRDE-1 foci.Additionally, we did not identify any changes of GFP::KIN-3 localization in tofu-4 mutants (Supplementary Fig. 2d, e)." 10.In the phospho mimic mutant that was made both serines were mutated.However, there is no data to show that this is the relevant situation.Single mutations should be tested before anything can be concluded.The double mimic could in some unforeseen way erase the effect of single events.Is the phospho mimic in PRDE-1 foci?
As suggested by the reviewer, we tested the single mutations.The S131D and S229D lesions each caused partial de-silencing individually.
We also examined the tofu-4 phospho mimetic mutant.We found that like the wild type protein, the mutant protein is still localized in the PRDE-1 foci.However, the PRDE-1 foci are a little smaller.The results are included in the main Figure 5e and in the supplementary Figure 4b.

Minor:
Please annotate the pachytene zone in 1A,B.For non-elegans researchers the presented information is too scarce.
As suggested by the reviewer, we have annotated the pachytene zone in 1A, B. The results have been included in the main Figure 1a and b.

Please provide reference for line2h and turmoil1 activation in piRNA mutants (page 5, bottom)
We have provided reference 8 for line2h and turmoil1 activation in piRNA mutants in the paper.
Please show the recombinant proteins purified from E. coli.The readers need to know about the quality of the protein preps used.
As suggested by the reviewer, the recombinant proteins purified from E. coli are shown in supplementary Figure 3a.

In the IP-Westerns, please provide information about quantities (how much was used for input in relation to the IP samples?)
We have provided this information in the main Figure 4a and 5b.
Testing CK2 activity in extracts seems a bit of a crude assay, given that many more kinases exist.Can a control be provided using extract from a kin-3 mutant (using the AID system that the authors developed)?
We provided the control using the extract from kin-3 mutant, this has been included in supplementary Figure 4c.As suggested by the reviewer, we have changed the sentences "In the C. elegans pachytene germline PRDE-1 foci colocalize with large regions of chromosome IV where the majority of piRNA genes reside and become positioned near the nuclear periphery 9 .The transcription of piRNA genes is thought to occur within chromatin marked by H3K27me3, a mark also found in telomeric chromatin 10 .It will be interesting to learn whether CK2 phosphorylation of TOFU-4 promotes the colocalization of the huge megabase-scale clusters of piRNA genes perhaps to coordinate piRNA transcription with piRNA export and processing." Page 13: '…promotes the subnuclear localization…' It is unclear to me what is meant.Not much is known about the subnuclear localization of PRDE-1 foci, apart from that there are foci.So what can be 'promoted'?I giess the author aim at potential colocalization of these foci with nuclear pores?Please explain better what is meant.